This proposal continues to explore the role played by tetrahydropteridines in the mixed function oxygenase reactions of phenylalanine and tyrosine hydroxylase. During the course of these reactions one atom of molecular oxygen is inserted into the aromatic ring of phenylalanine or tyrosine with a synchronous conversion of the tetrahydropteridine to an "active" or "dihydroquinonoid" form of the pteridine. Pteridine reductase, in the presence of NADH, converts the dihydropteridine back to the tetrahydro level. With the aid of affinity chromatograhy, homogeneous reductases have been isolated in good yield (approximately 30%) from rat and sheep liver. Using similar techniques it is intended to isolate the two hydroxylases from liver and brain tissue. Typical protein characterization procedures such as: polyacrylamide electrophoresis, titration with dimethyl suberimidate, gel filtration, equilibrium dialysis, circular dichroism and specific amino acid labeling, currently being applied to the dihydropteridine reductase to give structural information, and determine the enzyme affinity for substrate and cofactor, are to be extended to the hydroxylases. Analogs of the amino acid substrates containing photoaffinity labels will be synthesized as probes of the hydroxylase active sites, and in addition, previously synthesized fluorescent folate analogs will be used to investigate the pteridine binding site of the reductase. Improved assay procedures for the enzymatic reactions using fluorescent and fast-reaction techniques are described, together with chemical and kinetic experiments designed to determine whether the tetrahydropteridine cofactor participates in the coupled enzymatic reaction as an oxygen/hydroxyl carrier or merely as a reducing agent for the hydroxylases.